Composition and methods for enhancing receptor-mediated cellular internalization

ABSTRACT

Compositions and methods for improving cellular internalization of one or more compounds are disclosed. The compositions include a compound to be delivered and a biocompatible viscous material, such as a hydrogel, lipogel, or highly viscous sol. The composition also include, or are administered in conjunction with, an enhancer in an amount effective to maximize expression of or binding to receptors and enhance RME of the compound into the cells. This leads to high transport rates of compounds to be delivered across cell membranes, facilitating more efficient delivery of drugs and diagnostic agents. Compositions are applied topically orally, nasally, vaginally, rectally, and ocularly. The enhancer is administered with the composition or separately, either systemically or preferably locally. The compound to be delivered can also be the enhancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a reissue of U.S. Ser. No. 10/120,940, filed Apr.10, 2002, now U.S. Pat. No. 6,652,873, which is a continuation of priorpending application U.S. Ser. No. 09/412,821, filed Oct. 5, 1999, nowU.S. Pat. No. 6,387,390 which claims priority to U.S. provisionalapplication Ser. No. 60/103,117, filed Oct. 5, 1998, and which is acontinuation-in-part of U.S. Ser. No. 08/810,275, filed Mar. 3, 1997,now U.S. Pat. No. 5,985,320, which claims priority to U.S. provisionalapplication Ser. No. 60/012,721, filed Mar. 4, 1996 .

GOVERNMENT SUPPORT

This invention was made with government support under Hatch Act ProjectNo. PEN03466, awarded by the United States Department of Agriculture(USDA). The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The compositions and methods of use described herein generally are inthe field of materials and methods for enhancing cellularinternalization.

It is often difficult to deliver compounds, such as proteins, peptides,genetic material, and other drugs and diagnostic compoundsintracellularly because cell membranes often resist the passage of thesecompounds. Various methods have been developed to administer agentsintracellularly. For example, genetic material has been administeredinto cells in vivo, in vitro, and ex vivo using viral vectors, DNA/lipidcomplexes, and liposomes. While viral vectors are efficient, questionsremain regarding the safety of a live vector and the development of animmune response following repeated administration. Lipid complexes andliposomes appear less effective at transfecting DNA into the nucleus ofthe cell and potentially may be destroyed by macrophages in vivo.

Proteins and peptides are typically administered by parenteraladministration, or, in some cases, across the nasal mucous membrane.Uptake of drugs administered topically is frequently poor, anddegradation frequently occurs when drugs are administered orally. Forexample, hormones such as gonadotropin releasing hormone (“GnRH”) andits analogs have been administered to humans in an attempt to increasefertility by increasing systemic levels of luteinizing hormone (“LH”).When given often, low doses of native GnRH have been shown to inducefollicular development and ovulation. These drugs are typicallyadministered via an indwelling catheter into the abdominal cavity. Anexternal pump is attached to the catheter which injects the peptide atfrequent intervals. This method of administration is extremely invasiveand undesirable. Also, the method is prohibitively expensive for use inanimals.

It has recently been demonstrated that, by embedding individual cellpopulations in hydrogel media of macroscopic viscosity similar to thatcharacteristic of cell cytoskeleta, the rate of receptor-mediatedendocytosis can be significantly enhanced (Edwards, et al., Proc. Natl.Acad. Sci. U.S.A. 93:1786-91 (1996); PCT US97/03276 by MassachusettsInstitute of Technology and Pennsylvania State University Foundation).This enhancement effect appears to reflect a fluid-mechanical origin ofreceptor-mediated endocytosis, involving the rapid expansion of plasmamembrane in the vicinity of a receptor cluster leading to aninvaginating membrane motion that is sensitive to the viscous propertiesof the extracellular environment (Edwards, et al., Proc. Natl. Acad.Sci. U.S.A. 93:1786-91 (1996); Edwards, et al., Biophys. J 71:1208-14(1996)).

It has been found, however, that the delivery of compounds via areceptor-mediated route into the systemic circulation by noninvasivelydelivering the compound in a “rheologically-optimized” hydrogel may beinconsistent or poorly reproducible. It would be advantageous to betterunderstand the role of RME in uptake of compounds in order to developimproved methods of delivery of compounds, such as drugs,intracellularly.

The binding of ligands or assembly proteins to surface receptors ofeucaryotic cell membranes has been extensively studied in an effort todevelop better ways to promote or enhance cellular uptake. For example,binding of ligands or proteins has been reported to initiate oraccompany a cascade of nonequilibrium phenomena culminating in thecellular invagination of membrane complexes within clathrin-coatedvesicles (Goldstein, et al., Ann. Rev. Cell Biol. 1:1-39 (1985); Rodman,et al., Curr. Op. Cell Biol. 2:664-72 (1990); Trowbridge, Curr. Op. CellBiol. 3:634-41 (1991); Smythe, et al., J. Cell Biol. 108:843-53 (1989);Smythe, et al., Cell Biol. 119:1163-71 (1992); and Schmid, Curr. Op.Cell Biol. 5:621-27 (1993)). This process has been referred to asreceptor-mediated endocytosis (“RME”). Beyond playing a central role incellular lipid trafficking (Pagano, Curr. Op. Cell Biol. 2:652-63(1990)), RME is the primary means by which macromolecules entereucaryotic cells.

An effective strategy for enhancing the uptake of cytotoxic andtherapeutic drugs involves exploiting the rapidity and specificity oftransmembrane transport via receptor-mediated endocytosis (Goldstein, etal., Ann. Rev. Cell Biol. 1:1-39 (1985)) by targeting receptors on theplasma membranes of endothelial (Barzu, et al., Biochem. J. 15;238(3):847-854 (1986); Magnusson & Berg, Biochem. J. 257:65-56 (1989)),phagocytic (Wright & Detmers, “Receptor-mediated phagocytosis” in TheLung: Scientific Foundations (Crystal, et al., eds.), pp. 539-49 (RavensPress, Ltd., New York, N.Y. (1991)); and tumor cells, as well as cellsof other tissues. Receptor targeting has, however, not been championedas a means of avoiding intravenous injection of hard-to-absorbmacromolecules, probably because macromolecules often degrade prior toreaching receptors in the gastrointestinal tract following oraladministration, and do not appear to require receptor-mediation topermeate across the alveolar epithelium following inhalation. Othernoninvasive macromolecular drug delivery strategies either do not exposereceptors to the topical environment, for example transdermal delivery,or have been less extensively explored, such as nasal delivery (Illum,et al., Int. J. Pharm. 39:189-99 (1987)), vaginal delivery, or oculardelivery.

It is therefore an object of the present invention to providecompositions and methods for enhancing intracellular delivery ofbioactive and/or diagnostic agents, especially steroidal compounds andmaterials which are endocytosed by a receptor-mediated mechanism.

SUMMARY OF THE INVENTION

Compositions and methods for improving cellular internalization of oneor more compounds using a receptor mediated mechanism are disclosed. Thecompositions include a compound to be delivered and a biocompatibleviscous material, such as a hydrogel, lipogel, or highly viscous sol,and are administered subsequent to or with steroid or other materialbinding to the receptor at the site of application to enhance uptake(referred to as an “enhancer”). By controlling the apparent viscosity ofthe viscous materials, the rates of endocytosis, including nonspecific“pinocytosis” and specific RME, are increased. The rate of endocyticinternalization is increased when the ratio of the apparent viscositiesof cytosolic and extracellular media approaches unity. The compositionincludes, or is co-administered with, the enhancer, usually a steroid orother molecule binding to receptors at the site of application in anamount effective to maximize binding to the receptors or expression ofreceptors and enhance RME of the compound into the cells. This leads tohigh transport rates of compounds to be delivered across cell membranes,facilitating more efficient delivery of drugs and diagnostic agents.

Preferred viscous materials are hydrogels, lipogels (gels withnonaqueous fluid interstices) and highly viscous sols. The apparentviscosity of the composition is controlled such that it lies in therange of between 0.1 and 2000 Poise, preferably between 7 and 1000Poise, and most preferably between 2 and 200 Poise. Compounds to bedelivered include those that can be attached, covalently ornoncovalently, to a molecule that either stimulates RME or pinocytosisby binding to receptors on the plasma membrane, binds specifically toreceptors that undergo RME or pinocytosis independently of this binding(i.e., which are themselves “enhancers”) or at least can be associatedchemically or physically with other molecules or “carriers” thatthemselves undergo RME or pinocytosis. Exemplary compounds to bedelivered include proteins and peptides, nucleotide molecules,saccharides and polysaccharides, synthetic chemotherapeutic agents, anddiagnostic compounds. The examples demonstrate the roles of estrogen andprogesterone in vaginal delivery of peptide hormones. Peptide transportinto the systemic circulation is strongly steroid-dependent, with mostefficient transport of reproductive hormones occurring after estradioland progesterone pretreatment, when hormone receptors are maximallyexpressed. Preferred steroids include steroidal hormones such asestrogen and progesterone and glucocorticoids.

The compositions are applied to cell membranes to achieve high rates oftransport of the compound to be delivered across those membranes,relative to when non-viscous fluids are used with the enhancers or theviscous fluids are used alone. Compositions are applied topicallyorally, nasally, vaginally, rectally, and ocularly. The enhancer isadministered systemically or, more preferably, locally. Compositions canbe applied by injection via catheter, intramuscularly, subcutaneously,and intraperitoneally. Compositions can also be administered to thepulmonary or respiratory system, most preferably in an aerosol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b are graphs showing serum responses to iv injection andvacinal administration of vasopressin and leuprolide acetate. FIG. 8ashows serum cortisol response to iv injection and vaginal administrationof vasopressin. Vasopressin was injected intravenously (5 g dose)through a jugular catheter. The peptide was delivered vaginally in 5 mlof aqueous solution (200 g does). Standard errors are based on n=6. FIG.8b shows Serum LH response to iv injection and vaginal administration ofleuprole acetate (“LHRH analog”). LHRH analog was injected intravenously(5 g dose) into through a jugular catheter. The peptide was deliveredvaginally in 5 ml of aqueous solution (200 g dose). Standard errors arebased on n=6.

FIG. 2 is a graph showing bioavailability of LHRH analog followingvaginal administration as a function of methyl cellulose (“methocel”)concentration. Bioavailability is determined relative to intravenousinjection (FIG. 1b) and is based on LH response. The administered doseof LHRH analog was 200 g in 5 ml of methocel solution. Results are basedon animals that responded to vaginal treatment, with standard errorcomputed on the basis of n>4. In all cases, less than 50% of treatedanimals responded with LH levels greater than 3 ng/ml for more than onesampling point, with sampling times of 0, 30, 60, 90, 120, 180, 240,360, and 480 min.

FIG. 3 is a graph showing the percent of responding animals to LHRHanalog vaginal delivery as a function of (simulated) stage of theestrous cycle. Stage of estrous cycle was simulated by deliveringestradiol for two weeks to ovariectomized ewes (anestrus phase),followed by two weeks of delivery of estradiol and progesterone(mid-luteal phase), followed by a period of 48 h after progesteronewithdrawal (follicular phase). In each simulated phase, 10, 40, or 200mg of LHRH analog were delivered vaginally in 5 ml of aqueous ormethocel solution to groups of six ewes. Responding animals were definedas those treated animals with LH serum values exceeding 3 ng/ml for twoor more sampling points, with sampling times of 0, 30, 60, 60, 120, 180,240, 360, and 480 min.

FIG. 4 is a graph showing serum LH response to vaginal administration ofLHRH analog. LHRH analog was administered vaginally in ovariectomizedewes during the simulated mid-luteal phase in 5 ml of aqueous ormethocel (1.75% methyl cellulose) solution (40 μg dose). Standard errorsare based on n—6.

FIG. 5 is a graph of plasma LH concentration versus day of DEStreatment.

FIG. 6 is a graph of plasma LH concentration versus time followingadministration of DES, in combination with progesterone alone orprogesterone and estradiol.

FIG. 7 is a graph of percentage of maximum LH response versus day of DEStreatment for progesterone-primed ewes.

FIG. 8 is a graph of basal plasma LH concentration versus day of DEStreatment for progesterone-primed ewes.

FIG. 9 is a graph of percentage of maximum LH response versus day of DEStreatment for progesterone-primed ewes.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods for intracellular delivery of compounds in aviscous solution enhancing uptake are described. Cellularinternalization is enhanced (1) by increasing the rate ofreceptor-mediated endocytosis by controlling the viscosity of thesolution containing the compound to be delivered and (2) byco-administration of an enhancer (such as a steroid) in an amounteffective to maximize expression of or binding to receptors involved inendocytosis mediated uptake. The compositions include one or morebioactive or diagnostic compounds and a fluid with an apparent viscosityapproximately equal to the apparent viscosity of the cytosolic fluid inthe cell to which the composition is administered, and optionally, theenhancer. The enhancer can be delivered in the same formulation orseparately, before or after administration of the compounds to bedelivered to the site where they are to be delivered. Alternatively, thecompound can be administered in the viscous carrier solution at a timeselected to maximize relevant steroidal levels, for example,administered vaginally during estrus.

Preferably, the compound binds to or otherwise interacts with receptorson the surface of the cell to which it is to be delivered. If thecompound does not itself bind to or interact with receptors on the cellsurface, it can be administered in a viscous fluid that also includes acarrier for the compound. The carrier contains ligands that bind to orotherwise interact with cell surface receptors, which allows compoundsthat do not bind to or otherwise interact with cell surface receptors toparticipate in RME.

Compositions

The binding of ligands or assembly proteins to surface receptors ofeucaryotic cell membranes initiates or accompanies a cascade ofnonequilibrium phenomena culminating in the cellular invagination ofmembrane complexes within clathrin-coated vesicles. This process isknown as receptor-mediated endocytosis (RME). RME is the primary meansby which several types of bioactive molecules, particularlymacromolecules, enter eukaryotic cells.

Research by others has primarily focused on the identification andbiochemical characterization of the early and later stages of RME,ranging from formation of a clathrin coated pit to snap-off of a coatedvesicle. Determination of the compositions and methods forintracellularly administering compounds described herein involvedfocusing on a different aspect of RME, the process in which a membranedepression is initially formed at the outset of RME (i.e. the mechanismby which a spontaneous thrust of the cell membrane toward the cytosoloccurs). This process is referred to herein as the ‘nucleation stage’ ofRME. This terminology is intended to emphasize that the driving forcefor the spontaneous thrust of the membrane toward the cytosol is relatedto energy liberated by one or more of many possible exothermicmembrane-binding reactions, i.e., receptor-ligand binding, that precedeor accompany formation of a membrane depression.

Cell membranes are bound from without by extracellular fluid and fromwithin by cytosolic fluid. The inter- and extracellular fluids possessdifferent physical properties, such as density and fluid viscosity,whose values extend up to the membrane surface where they undergodiscontinuities. The membrane itself possesses unique equilibrium andnonequilibrium properties. An important property when consideringintracellular delivery is the membrane tension (the free energy of themembrane per unit surface area). Membrane tension is generally uniformand positive at an equilibrium membrane and can be measured by routinemicropipet experiments. Most reported membrane tension values have beengathered for red blood cells, and range from 4 dyne/cm to 0.01 dyne/cm.By contrast, the interfacial tension of an air/water interface is 73dyne/cm. Membrane tension can vary from point to point on the membranesurface as a consequence of various stimuli, such as non-uniform heatingof the membrane, membrane chemical reactions and membrane compositionalchanges. These variations can give rise to membrane and bulk-fluidmotion, termed Marangoni convection. This motion is characterized forthe most part by cytosolic and extracellular (apparent) viscosities.

Exothermic reactions can occur on the cell membrane, due toligand-receptor binding, adaptor-membrane binding, clathrin-membranebinding, a combination of these binding reactions, and other membranereactions. The exothermic reactions cause the membrane tension (energyper membrane area), at least momentarily, to be diminished at the pointwhere the reaction occurred. As the membrane tension is lowered, theconfigurational and intermolecular potential energies of membrane-boundmolecular complexes are also lowered.

The cell membrane tension is spatially nonuniform as a consequence ofthe exothermic reactions (i.e., membrane complex formation), resultingin membrane motion. This motion will possess a substantial componenttoward the cell cytosol so long as the cytosolic viscosity exceeds thatof the extracellular fluid.

This membrane motion causes membrane deformation, an event resisted bythe membrane tension. When the differences between the apparentviscosities of the cytosolic fluid and the extracellular fluid areextremely large, membrane deformation is strongly resisted and theinitial thrust of the membrane is damped. However, as the differencesbetween the apparent viscosities of the cytosolic fluid and theextra-cellular fluid become extremely small, membrane deformationbecomes progressively rapid.

Accordingly, the rate of endocytosis can be increased by adjusting theviscosity of the extracellular fluid so that it is approximately thesame as that of the cytosolic fluid, as described by PCT/US97/03276. Ifthe viscosity of the extra-cellular fluid is appreciably higher or lowerthan that of the cytosolic fluid, the rate of endocytosis decreases.This was shown experimentally in Example 1 and FIG. 3 of PCT US97/03276,in which the ratio of compounds that were internalized to thoseremaining on the surface (In/Sur) increased as the viscosity of theextracellular fluid increased, to a point at which the viscosityapproached that of the cytosolic fluid. Above that value, the ratiodecreased.

Clustering of membrane complexes is favorable for rapid internalization.The rate of internalization can be increased in proportion to themagnitude of binding energy. This is due, in part, to the specificity ofreceptors to particular ligands and/or adaptor proteins.

Clustering of complexes occurs in the vicinity of pits to which clathrintriskelions absorb from the cytosolic side of the cell membrane andsubsequently polymerize to form a clathrin coat. Some clustering hasalso been observed in the vicinity of caveolae, or non-clathrin-coatedpits. The membrane-tension depression occurring within the vicinity ofan evolving pit, originating in the process of membrane complexation, isdirectly proportional to the number of membrane complexes formed withinthat pit. In general, clustered complexes have been found to internalizesubstances more rapidly than nonclustered complexes.

The magnitudes of apparent viscosity difference and receptor clusteringhave each been found to alter the rate of RME. Membrane tension can alsobe manipulated to influence the rate of RME. Increasing the membranetension ‘hardens’ the cell membrane, making cell membrane depressionincreasingly prohibitive. This phenomenon has been commented upon bySheetz, M. P. and Dai, J. (1995), presented at the 60th Annual ColdSpring Harbor Symposium on Protein Kinases, Cold Spring Harbor, N.Y., onthe basis of studies that show an increased rate of endocytosis forneuronal growth cones coinciding with membrane tension lowering.

Accordingly, the rate of internalization can be increased by a)adjusting the viscosity of the extracellular fluid to approximate thatof the cytosolic fluid; b) forming complexes of the material to beinternalized; and c) reducing membrane tension. Compositions and methodsfor increasing the rate of endocytosis are described in detail below.

A. Viscous Hydrogels

Suitable viscous fluids for use in intracellularly administeringcompounds include biocompatible hydrogels, lipogels, and highly viscoussols.

A hydrogel is defined as a substance formed when an organic polymer(natural or synthetic) is cross-linked via covalent, ionic, or hydrogenbonds to create a three-dimensional open-lattice structure which entrapswater molecules to form a gel. Examples of materials which can be usedto form a hydrogel include polysaccharides, proteins and syntheticpolymers. Examples of polysaccharides include celluloses such as methylcellulose, dextrans, and alginate. Examples of proteins include gelatinand hyaluronic acid. Examples of synthetic polymers include bothbiodegradeable and non-degradeable polymers (although biodegradeablepolymers are preferred), such as polyvinyl alcohol, polyacrylamide,polyphosphazines, polyacrylates, polyethylene oxide, and polyalkyleneoxide block copolymers (“POLOXAMERS™”) such as PLURONICS™ or TETRONICS™(polyethylene oxide-polypropylene glycol block copolymers).

In general, these polymers are at least partially soluble in aqueoussolutions, such as water, buffered salt solutions, or aqueous alcoholsolutions. Several of these have charged side groups, or a monovalentionic salt thereof. Examples of polymers with acidic side groups thatcan be reacted with cations are polyphosphazenes, polyacrylic acids,poly(meth) acrylic acids, polyvinyl acetate, and sulfonated polymers,such as sulfonated polystyrene. Copolymers having acidic side groupsformed by reaction of acrylic or methacrylic acid and vinyl ethermonomers or polymers can also be used. Examples of acidic groups arecarboxylic acid groups, sulfonic acid groups, halogenated (preferablyfluorinated) alcohol groups, phenolic OH groups, and acidic OH groups.

Examples of polymers with basic side groups that can be reacted withanions are polyvinyl amines, polyvinyl pyridine, polyvinyl imidazole,polyvinylpyrrolidone and some imino substituted polyphosphazenes. Theammonium or quaternary salt of the polymers can also be formed from thebackbone nitrogens or pendant imino groups. Examples of basic sidegroups are amino and imino groups.

Alginate can be ionically cross-linked with divalent cations, in water,at room temperature, to form a hydrogel matrix. An aqueous solutioncontaining the compound to be delivered can be suspended in a solutionof a water soluble polymer, and the suspension can be formed intodroplets which are configured into discrete microcapsules by contactwith multivalent cations. Optionally, the surface of the microcapsulescan be crosslinked with polyamino acids to form a semipermeable membranearound the encapsulated materials.

The polyphosphazenes suitable for cross-linking have a majority of sidechain groups which are acidic and capable of forming salt bridges withdi- or trivalent cations. Examples of preferred acidic side groups arecarboxylic acid groups and sulfonic acid groups. Hydrolytically stablepoly-phosphazenes are formed of monomers having carboxylic acid sidegroups that are crosslinked by divalent or trivalent cations such asCa²⁺ or Al³⁺. Polymers can be synthesized that degrade by hydrolysis byincorporating monomers having imidazole, amino acid ester, or glycerolside groups. For example, a polyanionic poly[bis(carboxylatophenoxy)]phosphazene (PCPP) can be synthesized, which is crosslinked withdissolved multivalent cations in aqueous media at room temperature orbelow to form hydrogel matrices.

Methods for the synthesis of the polymers described above are known tothose skilled in the art. See, for example Concise Encyclopedia ofPolymer Science and Polymeric Amines and Ammonium Salts, (Goethals, ed.)(Pergamen Press, Elmsford, N.Y. 1980). Many of these polymers arecommercially available.

Preferred hydrogels include aqueous-filled polymer networks composed ofcelluloses such as methyl cellulose, dextrans, agarose, polyvinylalcohol, hyaluronic acid, polyacrylamide, polyethylene oxide andpolyoxyalkylene polymers (“poloxamers”), especially polyethyleneoxide-polypropylene glycol block copolymers, as described in U.S. Pat.No. 4,810,503. Several poloxamers are commercially available from BASFand from Wyandotte Chemical Corporation as “Pluronics”. They areavailable in average molecular weights of from about 1100 to about15,500.

As used herein, lipogels are gels with nonaqueous fluid interstices.Examples of lipogels include natural and synthetic lecithins in organicsolvents to which a small amount of water is added. The organic solventsinclude linear and cyclic hydrocarbons, esters of fatty acids andcertain amines (Scartazzini et al. Phys. Chem. 92:829-33 (1988)).

As defined herein, a sol is a colloidal solution consisting of a liquiddispersion medium and a colloidal substance which is distributedthroughout the dispersion medium. A highly viscous sol is a sol with aviscosity between approximately 0.1 and 2000 Poise.

Other useful viscous fluids include gelatin and concentrated sugar (suchas sorbitol) solutions with a viscosity between approximately 0.1 and2000 Poise.

The apparent viscosity of the extracellular fluid (the composition) mustbe approximately equal to the viscosity of the cytosolic fluid in thecell to which the compounds are to be administered. One of skill in theart can readily determine or reasonably estimate of the viscosity of thecytosolic fluid using a viscometer and measuring the applied stressdivided by measured strain rate at the applied stress that correspondsto the stress the cell membrane imparts upon the cytosolic andextracellular fluids during endocytosis. Methods for measuring thecytosolic viscosity include micropipette methods (Evans & Young,Biophys. J., 56:151-160 (1989)) and methods involving the motion ofmembrane-linked colloids (Wang et al., Science, 260:1124-26 (1993).Typical cytosol viscosities, measured by these techniques, range fromapproximately 50-200 Poise. Once this value is measured, the viscosityof the composition can be adjusted to be roughly equal to thatviscosity, particularly when measured via routine methods at the appliedstress that corresponds to the stress the cell membrane imparts upon thecytosolic and extracellular fluids during endocytosis.

The viscosity can be controlled via any suitable method known to thoseof skill in the art. The method for obtaining a viscous composition withthe desired apparent viscosity is not particularly limited since it isthe value of the apparent viscosity relative to the target cells whichis critical. The apparent viscosity can be controlled by adjusting thesolvent (i.e., water) content, types of materials, ionic strength, pH,temperature, polymer or polysaccharide chemistry performed on thematerials, and/or external electric, ultrasound, or magnetic fields,among other parameters.

The apparent viscosity of the compositions is controlled such that itlies in the range of between 0.1 and 2000 Poise, preferably between 7and 1000 Poise, and most preferably between 2 and 200 Poise. Theapparent viscosity can be measured by a standard rheometer using anapplied stress range of between 1 and 1000 Pascals, preferably between 1and 500 Pascals, and most preferably between 1 and 100 Pascals. Further,the viscosity of the compositions is controlled so that the quotient of(apparent viscosity of the cytosol of the target cells—apparentviscosity of the composition) and the apparent viscosity of the cytosolof the target cells is between approximately −0.1 and 0.3, preferablybetween approximately 0 and 0.3, more preferably between approximately 0and 0. 1, and most preferably between approximately 0 and 0.05.

The composition can be administered as an only slightly viscousformulation that becomes more viscous in response to a condition in thebody, such as body temperature or a physiological stimulus, like calciumions or pH, or in response to an externally applied condition, such asultrasound or electric or magnetic fields. An example is a temperaturesensitive poloxamer which increases in viscosity at body temperature.

The following are examples of suitable concentration ranges: Methocelsolutions in the range of between 1.0 and 2.0% (w/w), polyvinyl alcoholsolutions between 5 and 15%, pluronic acid solutions between 15 and 20%and trehalose solutions between 1 and 5%.

B. Enhancers

Compounds that can be attached, covalently or noncovalently. to amolecule that either stimulates receptor-mediated endocytosis (RME) orpinocytosis by binding to receptors on the plasma membrane, bindsspecifically to receptors that undergo RME or pinocytosis independentlyof this binding, or at least can be associated chemically or physicallywith other molecules or “carriers” that themselves undergo RME orpinocytosis, are referred to as enhancers for intracellular delivery.Examples include steroids such as estradiol and progesterone, and someglucocorticoids. Glucocorticocoids such as dexamethasone, cortisone,hydrocortisone, prednisone, and others are routinely administered orallyor by injection. Other glucocorticoids include beclomethasone,dipropianate, betamethasone, flunisolide, methyl prednisone, paramethasone, prednisolone, triamcinolome, alclometasone, amcinonide,clobetasol, fludrocortisone, diflurosone diacetate, fluocinoloneacetonide, fluoromethalone, flurandrenolide, halcinonide, medrysone, andmometasone, and pharmaceutically acceptable salts and mixtures thereofOther compounds also bind specifically to receptors on cell surfaces.Many hormone specific receptors are known. These can all be used toenhance uptake. Selection of molecules binding to receptors which arepredominantly found on a particular cell type or which are specific to aparticular cell type can be used to impart selectivity of uptake.

The enhancer is preferably administered at a time and in an amounteffective to maximize expression of receptors, and consequently receptormediated internalization of the compound. The enhancer can itself be thecompound to be delivered.

C. Compounds to be Delivered

As noted above, the compound to be delivered may be the same as ordifferent from the enhancer. The enhancer can be administered as part ofthe formulation containing the compound to be delivered or prior to oras part of a different formulation. The enhancer may be administeredsystemically, followed by administration of the compound to be delivereddirectly to the site where uptake is to occur.

Compounds to be delivered include proteins and peptides, nucleic acidmolecules including DNA, RNA, antisense oligonucleotides, triplexforming materials, ribozymes, and guide sequences for ribozymes,carbohydrates and polysaccharides, lipids, and other synthetic organicand inorganic molecules. Preferred bioactive compounds include growthfactors, antigens, antibodies or antibody fragments, and genes such asgenes useful for treatment of cystic fibrosis, A1A deficiency and othergenetic deficiencies.

Preferred hormones includes peptide-releasing hormones such as insulin,luteinizing hormone releasing hormone (“LHRH”), gonadotropin releasinghormone (“GnRH”), deslorelin and leuprolide acetate, oxytocin,vasoactive intestinal peptide (VIP), glucagon, parathyroid hormone(PTH), thyroid stimulating hormone, follicle stimulating hormone, growthfactors such as nerve growth factor (NGF), epidermal growth factor(EGF), vascular endothelial growth factor (VEGF), insulin-like growthfactors (IGF-I and IGF-II), fibroblast growth factors (FGFs),platelet-derived endothelial cell growth factor (PDECGF), transforminggrowth factor beta (TGF-β), and keratinocyte growth factor (KGF). Othermaterials which can be delivered include cytokines such as tumornecrosis factors (TFN-α and TNF-β), colony stimulating factors (CSFs),interleukin-2, gamma interferon, consensus interferon, alphainterferons, beta interferon; attachment peptides such as RGD; bioactivepeptides such as renin inhibitory peptides, vasopressin, detirelix,somatostatin, and vasoactive intestinal peptide; coagulation inhibitorssuch as aprotinin, heparin, and hirudin; enzymes such as superoxidedismutase, neutral endopeptidase, catalase, albumin, calcitonin,alpha-1-antitrypsin (A1A), deoxyribonuclease (DNAase), lectins such asconcanavalin A, and analogues thereof.

Diagnostic agents can also be delivered. These can be administered aloneor coupled to one or more bioactive compounds as described above. Theagents can be radiolabelled, fluorescently labeled, enzymaticallylabeled and/or include magnetic compounds and other materials that canbe detected using x-rays, ultrasound, magnetic resonance imaging(“MRI”), computed tomography (“CT”), or fluoroscopy.

D. Carriers for Compounds to be Delivered

The compounds to be delivered and/or enhancers can optionally beincorporated into carriers, which are then dispersed in a viscous fluidwith an apparent viscosity approximately equal to the cytosolic fluid ofthe cell to which the compounds are to be delivered. Exemplary carriersinclude viruses, liposomes, lipid/DNA complexes, micelles, protein/lipidcomplexes, and polymeric nanoparticles or microparticles.

The carrier must be small enough to be effectively endocytosed. Suitablecarriers possess a characteristic dimension of less than about 200 nm,preferably less than about 100 nm, and more preferably, are less thanabout 60 nm.

The carrier must be able to bind to a cell surface receptor. If thecarrier does not naturally bind, it is well known in the art how tomodify carriers such that they are bound, ionically or covalently, to aligand (i.e., LHRH) that binds to a cell surface receptor. For example,U.S. Pat. No. 5,258,499 to Konigsberg et al. describes the incorporationof receptor specific ligands into liposomes, which are then used totarget receptors on the cell surface.

The use of carriers can be important when the compound to be delivereddoes not bind to or otherwise interact with cell surface receptors. Thecompound can be incorporated into a carrier which contains a ligand orother moiety which binds to or interacts with cell surface receptors.Then, due to the binding of or interaction with the receptor to the cellsurface and the apparent viscosity of the composition, the carrier (andencapsulated compound) is intracellularly delivered by endocytosis.

The use of carriers can be particularly important for intracellularlydelivering nucleic acid molecules. In one embodiment, nucleic acidmolecules are encapsulated in a liposome, preferably a cationicliposome, that has a receptor-binding ligand, such as LHRH, on itssurface. The liposome is then dispersed in a viscous fluid. When thecomposition is administered, the liposomes are endocytosed by the cell,and the nucleic acid molecules are released from the liposome inside thecell.

E. Compositions for Lowering or Raising Membrane Tension

The efficiency of the method can be increased by lowering the membranetension. Suitable methods for lowering membrane tension includeincluding a biocompatible surface active agent in the hydrogel,performing exothermic reactions on the cell surface (i.e., complexformation), and applying an external field to the cell surface. Suitablebiocompatible surface active agents include surfactin, trehalose, fattyacids such as palmitin and oleic acid, polyethylene glycol, hexadecanol,and phospholipids such as phosphatidylcholines andphosphatidylglycerols. Suitable complex-forming chemical reactionsinclude the reaction of receptor-binding ligands with cell surfacereceptors for these ligands, exothermic reactions such as occur betweensodium salicylate and salicylic acid, and neutralization reactions asbetween hydrochloric acid and ammonia (Edwards et al. 1996 Biophys. J.71, 1208-1214). External fields that can be applied to a cell surface toreduce membrane tension include ultrasound, electric fields, and focusedlight beams, such as laser beams.

The rate of cellular internalization can also be increased by causingthe clustering of receptors on the cell membrane. This can beaccomplished, for example, by creating zones on the membrane where themembrane tension is relatively high, causing the membrane fluid to flowtoward the zone of high membrane tension. This flow can carry receptorslocalized in the membrane toward each other, causing them to cluster.

Methods of Administration

In a preferred embodiment, the compound to be delivered and/or theenhancer are contained in the same formulation for simultaneousadministration. Alternatively, the composition and steroid are providedas parts of a kit, for separate administration. As shown in theexamples, the enhancer may be a hormone such as estradiol orprogesterone, administered systemically, while the compound to bedelivered is administered topically at a site where delivery is enhancedby the hormone, such as the vaginal mucosa.

The compositions can be applied topically to the vagina, rectum, nose,eye, ear, mouth and the respiratory or pulmonary system. Preferably, thecompositions are applied directly to the cells to which the compound isto be delivered, usually in a topical formulation. The enhancer can beadministered simultaneously with or after administration of thecomposition including the viscous gel and agent to be delivered. Theadministration schedule (e.g., the interval of time betweenadministering the enhancer and administering the gel composition) can bereadily selected by one of skill in the art to maximize receptorexpression and/or binding before exposure of the cell surface to theagent to be delivered.

The compositions are particularly advantageous for gene delivery andhormone therapy. By delivering a composition containing peptides such asGNRH or its analogues across the vaginal or nasal membranes, thecompositions can be used to treat a variety of human hormone-baseddisorders.

The dosage will be expected to vary depending on several factors,including the patient, the particular bioactive compound to bedelivered, and the nature of the condition to be treated, among otherfactors. One of skill in the art can readily determine an effectiveamount of the bioactive compound or compounds to administer to a patientin need thereof.

The method involves administering the composition to cells to enhancethe rate of transport across the cell membranes, relative to the rate ofdelivery when non-viscous fluids are used in combination with enhanceror when viscous fluids are used without enhancer. Examples of methods ofadministration include oral administration, as in a liquid formulationor within solid foods, topical administration to the skin or the surfaceof the eye, intravaginal administration, rectal administration,intranasal administration, and administration via inhalation. When thecomposition is administered orally or by inhalation, it is preferredthat it is administered as a dry powder that includes a swellablehydrogel that is designed to swell to an appropriate viscosity afterdelivery to the desired location. After inhalation, for example, thehydrogel absorbs water to obtain the desired viscosity and then deliversagents to the respiratory system. When administered orally, a hydrogelcan be selected that does not absorb water under conditions present inthe upper tract, but which does absorb water under conditions present inthe lower gastrointestinal tract (i.e., at a pH greater than about 6.5).Such hydrogels are well known to those of skill in the art. The use ofsuch compositions can optimize the delivery of agents to the lowergastrointestinal tract.

Applications for the Compositions and Methods

The methods and compositions described herein are useful in a variety oftherapeutic and diagnostic applications for humans and other animals.Preferred applications include the treatment of infertility and disease,such as cancer. The compositions can be used in various hormonereplacement therapies as well. In a preferred method of use, viscouscompositions are used to deliver progesterone vaginally to inducesecretory transformation of the endometrium and promote development ofpregnancy.

The compositions and methods of use thereof described herein will bemore clearly understood with reference to the following non-limitingexamples.

EXAMPLE 1 Peptide Transport Across the Vaginal Epithelium of Sheep

This study was intended to examine the relevance of control of theapparent viscosity of the extracellular fluid/cytosolic fluid to theenhancement of peptide drug delivery into the body via a noninvasiveroute by examining peptide transport across the vaginal epithelium ofsheep.

A. Receptor-mediated Transport of a Peptide, No Exogenous Steroid.

First, a peptide that undergoes receptor-mediated transport across thevaginal epithelium was identified by studying the permeation of peptidesof varying molecular weight in a sheep model. Peptides were deliveredvaginally to sheep in 5 ml of aqueous or methocel solutions with typicalpeptide concentrations of 10-40 μg/ml. A group of 18 intact ewes wereutilized for these experiments. For each study, sheep were randomlyassigned to a treatment group. In GnRH studies where each animalreceived all possible treatment combinations, each animal was assignedto an initial treatment group at random and subsequently randomly toeach of the remaining treatment groups. A minimum of 5 days (typically10 or more days) was allowed between experiments on a given animal, toprovide sufficient time for complete recovery of pituitaryresponsiveness to the highest doses of the GnRH agonist used. A 16G 150mm jugular catheter (Abbocath-T, Abbott Laboratories, Chicago, Ill.) wasinserted and blood samples collected at 0, 30, 60, 90, 120, 180, 240,360, 480 and 1440 min. after treatment. Luteinizing hormone (LH) levelswere determined.

The bioavailabilities of vasopressin (1084 Da), salmon calcitonin (3416Da), and insulin (5786 Da) all were found to be less than 0.1% followingvaginal administration in an aqueous buffer. Leuprolide acetate[luteinizing hormone releasing hormone (LHRH) analog] (1209 Da),however, exhibited high bioavailability (2.6±0.9%) based on biologicalresponse, even though its molecular weight is slightly larger than thatof vasopressin. A comparison of the biological response to vasopressinand leuprolide acetate is shown in FIGS. 1a and 1b. Vasopressinadministered by intravenous injection leads to high systemic cortisollevels within the first hour following treatment. However no detectablechange in systemic cortisol levels was observed following vaginaladministration (FIG. 1a). In contrast, LHRH analog produced significantluteinizing hormone (LH) response following intravenous injection andfollowing vaginal administration (FIG. 1b). The near coincidence of peakserum LH concentrations following injection and vaginal administrationindicated rapid internalization of leuprolide acetate, characteristic ofa receptor-mediated route of transport.

B. Enhancement of Transport using a Viscous, Balanced Carrier.

LHRH analog was placed in methyl cellulose solutions (“methocels”) ofvarying apparent viscosity. Studies of transferrin-mediated endocytosison single cells have shown peak endocytosis rates at methyl celluloseconcentrations between 1.25 and 1.75%, at which concentrations themethocels exhibit apparent viscosities in a range typical ofintracellular viscosities (Evans & Yeung, Biophys.J. 56:151-60 (1989)).First, 200 μtg of leuprolide acetate in 5 ml of aqueous solutions withmethocel weight concentrations varying between 0% and 3.0% werevaginally administered. LHRH analog bioavailability was found toincrease as methocel concentration increased to 1.75%, then to fall athigher methocel concentration (FIG. 2), mirroring a trend observed forreceptormediated endocytosis with single cells (Edwards, et al., Proc.Natl. Acad. Sci. U.S.A. 93:1786-91 (1996)). This appears to suggest thattransfer of LHRH analog into the systemic circulation is rate-limited byendocytic transfer from the apical side of the vaginal epithelium, whichcan itself be controlled by the viscosity of the methocel solutionwithin which it is administered, for the fluidmechanical reasonsdescribed above and in Edwards, et al., Proc. Natl. Acad. Sci. U.S.A.93:1786-91 (1996).

The enhanced bioavailability of LHRH also coincides with a longer-termrelease into the systemic circulation at the optimal methocelconcentrations. This suggests the possibility of a diffusion-controlleddelivery process, rather than an active, endocytic-controlled process;that is, increasing hydrogel viscosity might be related to diminishedrate of peptide diffusion through the hydrogel to the vaginalepithelium. To test this hypothesis, the efficacy of a second,physically cross-linked hydrogel that was believed would not enhanceendocytosis, but whose apparent viscosity (in the range of hydrogelconcentrations 0.0-5.5%) was similar to that of the methocels (in therange 0.0-3.0%) was examined. It was anticipated that the physicallycross-linked structure of the “control” hydrogel would prevent itsdeformation with (and entry into) invaginating sites on the epithelialmembrane, hence impeding, rather than enhancing, endocytic uptake.

The results showed that when 5 ml of solution containing the physicallycross-linked control gel was administered vaginally, the bioavailabilityof LHRH analog diminished with increasing concentration of the hydrogelin the range of 0.0-5.5%. Importantly, the duration of LHRH analogdelivery also diminished with increasing control gel concentration,which is an unexpected effect if LHRH analog delivery ispassive-diffusion controlled.

To determine whether membrane damage might explain the results shown inFIG. 2, vasopressin was vaginally administered in methocel solutions of1.5 and 1.75%. Identical to the saline vaginal administration (FIG. 1a),no detectable changes in concentrations of cortisol were observed whenvasopressin was administered with the methocel solutions, indicatingthat the barrier properties of the membrane to passive transport remainintact.

C. Determination of Role of Steroids in Uptake and Transport

Biological response to vaginal LHRH analog administration exhibited abimodal distribution in the studies (see FIG. 2), with approximately 30%of animals showing little or no response at all. No such bimodalresponse was observed when LHRH analog was administered by intravenousinjection (FIG. 1b), indicating that the source of the bimodal responseresides in the vaginal absorption pathway. It was therefore hypothesizedthat the responsiveness of animals to LHRH analog vaginal deliveryvaried with steroid-dependent hormone receptor expression (estrouscycle). To test this hypothesis, a group of ewes was ovariectomized andadministered estradiol and progesterone to mimic the animals' estrouscycle. Ewes were pre-medicated with atropine (0.02 mg/lb) and Telazol(R) (2 mg/lb) intramuscularly. After induction of recumbency, thipental(5% in water) was administered intravenously to induce sufficientanesthesia to permit endotracheal intubation. Anesthesia was maintainedusing halothane in oxygen at 1-2 liters per minute. Ovaries were removedthrough a mid-line incision.

Within 24 h of surgery, a 1.5 cm silicone implant of estradiol(Compudose 200, Elanco, Ind.) was inserted into the left ear to providea basal level of estradiol. The anestrus state was simulated after twoweeks of estradiol delivery following surgery. Experiments wereperformed in the simulated anestrus state (i.e. two weeks after surgery)as described above. Immediately following the last blood sample, aprogesterone-releasing intravaginal device (CIDR-G, Carter Hold HarveyPlastic Products, Hamilton, New Zealand) was inserted.

In a parallel study, an alternative progesterone-releasing device(Snychro-Mate-B, Sanofi Animal Health, Overland Park, Kans.) was placedin the left ear. The mid-luteal phase was simulated after permitting a10 day intravaginal or ear progesterone treatment. Experiments wereperformed in the simulated mid-luteal phase as described above.

The progesterone-releasing (vaginal or ear) implant was removed and thefollicular phase was simulated by allowing a time lapse of 48 h.Experiments were performed in the follicular phase as described above.

Next, 200 μg of leuprolide acetate in 5 ml of aqueous solutions wasvaginally administered during simulated anestrus (estradiol only),mid-luteal (estradiol and progesterone), and follicular (48 h afterprogesterone withdrawal) phases, as described above. When LHRH analogwas delivered in aqueous solutions with or without 1.75% methocel, itwas found that less than 50% of animals responded during estrogenreplacement without progesterone. In contrast, 100% of animals respondedwhen treated with progesterone and estradiol (FIG. 3). This same trendalso was observed for the other LHRH analog doses. That is, areproducible response was observed in all animals only duringprogesterone and estradiol treatment, when the animals can be expectedto express maximal numbers of hormone receptors.

This confirms the hypothesis that the LH response of animals depended onsteroid milieu, as is consistent with the hypothesis of uptake of LHRHanalog occurs via a receptor-mediated route.

Three doses of LHRH analog: 10, 40, and 100 μg, were administered. Itwas found that during progesterone and estradiol treatment, the highestand lowest doses resulted in LH responses that were either saturated(maximal LH response with and without methocel) or undetectable,presumably due to the sigmoidal dose-response nature of LHRH analogtreatment. The results of the intermediate dose-response study are shownin FIG. 4. The 1.75% methocel administration results in abioavailability of 6% compared to 10% for the case of the 0% methocel.These results agree with the results of the uncontrolled animal study(FIG. 2) (minus non-responders).

A significant finding of this study is that LHRH analog delivery acrossthe vaginal mucosa is receptor-mediated, with a reproducibility that canbe increased by controlling the stage of the estrous cycle. Thisapproach to be peptide delivery can be further improved by controllingthe viscous properties of the medium that contacts the vaginal mucosa,and from which the peptide transfers. Unlike the single-cell systemswhere a similar phenomenon has been observed, the vaginal mucosaincludes a mucus barrier that is itself highly viscous and presumablycombines with the administered hydrogel to create a mixture ofartificial and physiological hydrogels whose net viscous properties actto control the rate of vesicle formation along the apical epithelialmembrane. While the exact nature of this mixed gel remains unclear (as,for example, the administration of estradiol and progesterone changesthe Theological properties of the mucus lining, potentially providing analternative interpretation of the results observed in FIG. 3), thathydrogel-enhancement of receptor-mediated transport can be achieved atvaginal mucosa suggests that a similar enhancement can achieved at otherdrug delivery sites, such as at the nasal mucosa.

The ability to enhance the delivery of LHRH analog into the systemiccirculation by delivering LHRH analog in a “rheologically-optimized”hydrogel should help to make noninvasive LHRH analog therapies (such asthe treatment of endometriosis or prostate cancer) more viable than atpresent. Recognition that it enters the body via a receptor-mediatedroute can further lead to hormonal-control strategies to minimizeirreproducibility. Finally, the chemical attachment of LHRH analog toother molecules or nanoparticulate carriers that are too large to crossepithelial barriers of the body at a therapeutically relevant rate yetsufficiently small to enter an endocytic vesicle should make it possibleto use LHRH analog as a kind of locomotive to propel other molecules,vesicle, or particles into the body without the need for injection.

EXAMPLE 2 Steroid and GNRH Transport

Studies were conducted to assess the involvement of steroids inmodulating the transport of GnRH across the vaginal mucosa. The mainobjectives were to confirm the need for steroids in vaginal GNRHtransport, to determine if treatment with both progesterone andestradiol were necessary, and to demonstrate down-regulation of LHsecretion with daily administration of GnRH agonist.

A. Chronic Vaginal Dosing of DES to Suppress LH Secretion

The objective was to determine if chronic vaginal dosing with 200 μg ofdeslorelin (“DES”) in gel would be able to suppress secretion of LH.Lower doses of DES will result in the down-regulation of the anteriorpituitary gland in sheep. Ovariectomized sheep were used for the study,since they secrete high levels of LH in the absence of ovarian steroids.The sheep were dosed daily with DES in 5 mL of gel, or gel only for 17days. A single blood sample was collected by jugular venipuncture.Plasma was collected and assayed for LH.

Results from this study are shown in FIG. 4. There were no differencesin the average concentrations between the DES and control groups overthe course of the study. LH secretion appears to have been slightlysuppressed between days 6 and 12 of the study. However, a much greaterinhibition in LH secretion was expected if significant amounts of DESwere crossing the vaginal mucosa. An earlier study demonstrated thatestrous cycles could be inhibited with daily vaginal administration ofGnRH agonist. In this study, however, all the animals had intactovaries. Previous studies have followed LH release after a singledosing. It has been shown that sheep treated with estradiol andprogesterone respond much better in terms of the percentage of animalsexhibiting LH release, the magnitude of the LH release, and a reductionin the variance of the response. Therefore, it is likely that treatmentwith progesterone, either alone or in combination with estradiol, wasrequired for the GNRH agonist to be transported across the vaginalmucosa.

B. Determination of Roles of Progesterone and Estradiol in VaginalTransport.

This study was conducted to determine if progesterone alone, orprogesterone in combination with estradiol, is required to ensure GnRHtransport across the vaginal epithelium. In past studies, estradiol wasgiven as an implant inserted in the outer ear, which is a common methodof administering estradiol to animals. However, it results in high andvariable estradiol concentrations in jugular blood. It would be helpfulto eliminate this steroid from the animal model, if possible, sincedelivering estradiol using GnRH-based technologies is of interest.

Ewes (n=12) were treated with progesterone using a vaginal CIDR device.In addition, six sheep received a 15 mm silastic implant of estradiol.Five days later, all sheep were treated vaginally with 200 μg of DES ingel. Blood samples were taken every 30 minutes for 2 hours followingtreatment and then every 60 minutes for an additional four hours.

All ewes in this experiment responded with a robust discharge of LHfollowing GnRH treatment. There was a distinct difference in the patternof LH release between the two groups (FIG. 13). The peak LH occurredearlier in ewes treated only with progesterone (120 minutes) than inewes treated with progesterone and estradiol (240 minutes).

The difference in the timing of the peak LH between the groups is nearlyidentical to those differences between ovariectomized and ovariectomizedplus estradiol treated ewes (see Deaver et al., Domest. Anim.Endocrinol. 4(2): 95-102 (1987)). Thus, it is likely that the differencein the patterns of LH release is attributable to estradiol's effects onpituitary responsiveness to GNRH and not the vaginal uptake mechanism.Furthermore, if the latter were correct, then the lag time betweentreatment and vaginal transfer of GnRH into the circulatory system wouldbe on the order of 70-80 minutes.

C. Effect of Vaginal Administration of DES in Progesterone-Primed Ewes.

The objective of this study was to determine if daily vaginaladministration of DES in progesterone-primed ewes would cause areduction in basal LH secretion and loss of pituitary responsiveness ofGnRH. Six ovariectomized ewes were used for the study. Vaginal CTDRdevices containing progesterone were inserted. Twenty-four hours later,ewes were dosed daily with 200 μg of DES (GnRH agonist) in 5 mL of gel.Each day, blood samples were collected at 0 and 120 minutespost-treatment. These times were selected in order to evaluate changesin basal secretion of LH (time 0) and the peak LH response followingGnRH administration (time 120).

The change in LH between 0 and 120 minutes was greatest in 3 of 6 eweson the first day of DES administration. In the fourth ewe, a robustrelease of LH was observed following the first DES treatment, but theincrease in LH was even higher following the second treatment with DES.When the differential in LH release following the first treatment withDES is assigned a value of 100% and the change in responsiveness plottedagainst time (FIG. 7), it is clear that continued daily treatmentsignificantly reduced pituitary responsiveness to DES. In addition,significant decreases in basal LH also occurred in these animals (FIG.8).

In the two remaining animals, the change in LH secretion continued toincrease 5 to 7 days following the initiation of DES treatment. However,once the maximum response was achieved, pituitary-responsiveness to DESrapidly declined (FIG. 16; Ewe 9). A reasonable interpretation of thesedata is that insufficient DES was transported over the first severaldays to initiate the down-regulation phenomena. However, once thetransport mechanism became optimized, adequate transport of DES wasachieved to down-regulate LH release from the anterior pituitary gland.

D. Direct Vaginal Administration of DES Enhanced Uptake.

An attempt was made to treat ewes with progesterone using the systemicadministration of a depot form of progesterone and ear implants of asynthetic progestogen. Pituitary release of LH was poor following thevaginal administration of DES in gel. When LH release in these animalswas not observed, the gel from the same preparations used vaginally wasinjected subcutaneously. LH release was then obtained, confirming thatthe DES/gel preparations contained biologically active material. Giventhe consistent responses obtained in earlier experiments and thoseobtained more recently using the CIDR delivery system, it was concludedthat the progesterone (generally) should be applied directly to thevagina in order to achieve sufficient local concentrations. Given thatluteal phase sheep also respond well, systemic administration of greateramounts of progesterone than used here, in formulations that willmaintain consistently high concentrations in the blood, should alsowork.

E. Controls Show Uptake is Selective.

Another study provided information about the role of the silastic CIDRdevice itself in the delivery process. The study was based on concernthat the CIDR might be damaging the vaginal mucosa, allowing for GNRHtransport by a non-selective mechanism. CIDR devices were inserted intosix ewes. Five days later, all ewes were treated vaginally with DES.Five of the six ewes had a robust release of LH. After the initialdosing, three ewes were treated with DES and three with gel alone everyday for 18 days. At the end of the 18-day treatment period, all eweswere again treated with DES in gel. This time, none of the ewesdisplayed a robust release of LH.

Review of the protocol and notes showed that the CIDR devices, whichwere designed to release progesterone only over a 10- to 12-day period,were not changed mid-way through the trial as initially planned.Consequently, by the time the second DES administration was given, theCIDR devices had been in place for approximately 24 days. The ewestherefore were no longer receiving adequate amounts of progesteronelocally to maintain the vaginal transport system. This study showed thatthe CIDR per se does not facilitate vaginal uptake of GnRH.

Conclusions

Based on the outcome of all experiments, local short periods ofprogesterone treatment activates a mechanism for transporting GnRHagonist across the vaginal mucosa in sufficient amounts to acutely causethe release of LH and the down-regulation of LH release with repeateddosing.

Local exposure of the vagina to progesterone is preferred for thetransport of GnRH agonist across the mucosal membrane. Systemicadministration of either progesterone or synthetic progestogens is notpreferred for achieving adequate priming of the vaginal mucosa for GnRHtransport. Intravaginal devices used for administration of progesteronedo not appear to directly effect vaginal transport of GnRH.

Approximately 50% of ewes will have significant transport after only 24hours of exposure to progesterone, and essentially 100% of the ewes willtransport GnRH after four days of exposure. Co-administration ofestradiol will alter the time course of LH release in progesteronetreated ewes, which likely is due to a direct effect on the anteriorpituitary gland. The lag time is about 70-80 minutes between vaginaladministration of GnRH and the transport of sufficient amounts of GnRHagonist into the blood to cause LH release. Chronic administration ofGnRH agonist across the vaginal mucosa will reduce the basal secretionof LH and down-regulate the ability of the anterior pituitary gland torespond to GnRH agonist.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The references citedherein are hereby incorporated by reference.

1. A composition comprising (i) a carrier selected from the groupconsisting of viruses, liposomes, lipid/DNA complexes, micelles,protein/lipid complexes, nanoparticles, and microparticles, wherein thecarrier comprises a hormone targeting the carder carrier to tissueexpressing a receptor for the hormone, and (ii) a viscous materialselected from the group consisting of hydrogels, lipogels and sols,wherein the viscous material alters cellular uptake of the carrier,wherein the viscous material has an apparent viscosity between 0.1 and2000 2 and 200 Poise at a sheer stress of between 1 and 1000 Pascal andat a strain rate approximately that of endocytosis, and wherein theapparent viscosity is approximately equal to the viscosity of thecytosolic fluid in the cell to which the hormone is to be delivered. 2.The composition of claim 1, wherein the carrier further comprises anactive agent.
 3. The composition of claim 1, wherein the tissue ismucosal tissue.
 4. The composition of claim 3, wherein the tissue islower gastrointestinal tract mucosal tissue.
 5. The composition of claim1, wherein the hormone is GnRH or an analog of GnRH.
 6. The compositionof claim 2, wherein the active agent is selected from the groupconsisting of proteins, peptides, nucleotide molecules, saccharides,polysaccharides, lipids, synthetic organic and inorganic molecules,synthetic chemotherapeutic agents, and diagnostic compounds.
 7. Thecomposition of claim 1, wherein the composition is in a form suitablefor administration to the vagina or rectum.
 8. The composition of claim1, wherein the composition is in a form suitable for administration tothe nose, eye, or mouth.
 9. The composition of claim 1, wherein thecomposition is in a form suitable for administration to the respiratoryor pulmonary system.
 10. The composition of claim 1, wherein thecomposition is in a form suitable for oral administration.
 11. Thecomposition of claim 1, wherein the composition is in a form suitablefor parenteral or systemic administration.
 12. The composition of claim1, wherein the viscous material is a hydrogel.
 13. The composition ofclaim 12, wherein the hydrogel comprises methyl cellulose.
 14. Thecomposition of claim 1, wherein the viscous material increases the rateof cellular internalization for the carrier.
 15. The composition ofclaim 1, wherein the carrier is a microparticle or nanoparticle.
 16. Thecomposition of claim 5, wherein the viscous material comprises methylcellulose.
 17. The composition of claim 16, wherein the methyl celluloseconcentration ranges from 1 to 2% (w/w).
 18. The composition of claim 1,wherein the hormone is a reproductive hormone.